Stenotrophomonas maltophilia OleC-Catalyzed ATP-Dependent Formation of Long-Chain Z-Olefins from 2-Alkyl-3-hydroxyalkanoic Acids.

The bacterial pathway of olefin biosynthesis starts with OleA catalyzed "head-to-head" condensation of two CoA-activated long-chain fatty acids to generate (R)-2-alkyl-3-ketoalkanoic acids. A subsequent OleD-catalyzed reduction generates (2R,3S)-2-alkyl-3-hydroxyalkanoic acids. We now show that the final step in the pathway is an OleC-catalyzed ATP-dependent decarboxylative dehydration to form the corresponding Z olefins. Higher kcat /Km values were seen for substrates with longer alkyl chains. All four stereoisomers of 2-hexyl-3-hydroxydecanoic acid were shown to be substrates, and GC-MS and NMR analyses confirmed that the product in each case was (Z)-pentadec-7-ene. LC-MS analysis supported the formation of AMP adduct as an intermediate. The enzymatic and stereochemical course of olefin biosynthesis from long-chain fatty acids by OleA, OleD and OleC is now established.

Elucidation of final steps of the marineosins biosynthetic pathway through identification and characterization of the corresponding gene cluster.

The marine Streptomyces sp. CNQ-617 produces two diastereomers, marineosins A and B. These are structurally related to alkyl prodiginines, but with a more complex cyclization and an unusual spiroaminal skeleton. We report the identification of the mar biosynthetic gene cluster and demonstrate production of marineosins through heterologous expression in a S. venezuelae host named JND2. The mar cluster shares the same gene organization and has high homology to the genes of the red cluster (which directs the biosynthesis of undecylprodiginine) but contains an additional gene, named marA. Replacement of marA in the JND2 strain leads to the accumulation of premarineosin, which is identical to marineosin with the exception that the middle pyrrole (Ring B) has not been reduced. The final step of the marineosin pathway is thus a MarA catalyzed reduction of this ring. Replacement of marG (a homologue of redG that directs undecylprodiginine cyclization to give streptorubin B) in the JND2 strain leads to the loss of all spiroaminal products and the accumulation of 23-hydroxyundecylprodiginine and a shunt product, 23-ketoundecylprodiginine. MarG thus catalyzes the penultimate step of the marineosin pathway catalyzing conversion of 23-hydroxyundecylprodiginine to premarineosin. The preceding steps of the biosynthetic marineosin pathway likely mirror that in the red-directed biosynthetic process, with the exception of the introduction of the hydroxyl functionality required for spiroaminal formation. This work presents the first experimentally supported scheme for biosynthesis of marineosin and provides a new biologically active molecule, premarineosin.

Stereospecific synthesis of 23-hydroxyundecylprodiginines and analogues and conversion to antimalarial premarineosins via a Rieske oxygenase catalyzed bicyclization.

Facile and highly efficient synthetic routes for the synthesis of (S)- and (R)-23-hydroxyundecylprodiginines ((23S)-2, and (23R)-2), 23-ketoundecylprodiginine (3), and deuterium-labeled 23-hydroxyundecylprodiginine ([23-d]-2) have been developed. We demonstrated a novel Rieske oxygenase MarG catalyzed stereoselective bicyclization of (23S)-2 to premarineosin A (4), a key step in the tailoring process of the biosynthesis of marineosins, using a marG heterologous expression system. The synthesis of various A-C-ring functionalized prodiginines 32-41 was achieved to investigate the substrate promiscuity of MarG. The two analogues 32 and 33 exhibit antimalarial and cytotoxic activities stronger than those of the marineosin intermediate 2, against Plasmodium falciparum strains (CQ(S)-D6, CQ(R)-Dd2, and 7G8) and hepatocellular HepG2 cancer cell line, respectively. Feeding of 34-36 to Streptomyces venezuelae expressing marG led to production of novel premarineosins, paving a way for the production of marineosin analogues via a combinatorial synthetic/biosynthetic approach. This study presents the first example of oxidative bicyclization mediated by a Rieske oxygenase.

Discovery and Optimization of Tambjamines as a Novel Class of Antileishmanial Agents.

Leishmaniasis is a neglected tropical disease that is estimated to afflict over 12 million people. Current drugs for leishmaniasis suffer from serious deficiencies, including toxicity, high cost, modest efficacy, primarily parenteral delivery, and emergence of widespread resistance. We have discovered and developed a natural product-inspired tambjamine chemotype, known to be effective against Plasmodium spp, as a novel class of antileishmanial agents. Herein, we report in vitro and in vivo antileishmanial activities, detailed structure-activity relationships, and metabolic/pharmacokinetic profiles of a large library of tambjamines. A number of tambjamines exhibited excellent potency against both Leishmania mexicana and Leishmania donovani parasites with good safety and metabolic profiles. Notably, tambjamine 110 offered excellent potency and provided partial protection to leishmania-infected mice at 40 and/or 60 mg/kg/10 days of oral treatment. This study presents the first account of antileishmanial activity in the tambjamine family and paves the way for the generation of new oral antileishmanial drugs.

Total Synthesis and Antimalarial Activity of 2-(p-Hydroxybenzyl)-prodigiosins, Isoheptylprodigiosin, and Geometric Isomers of Tambjamine MYP1 Isolated from Marine Bacteria.

Highly efficient and straightforward synthetic routes toward the first total synthesis of 2-(p-hydroxybenzyl)-prodigiosins (2-5), isoheptylprodigiosin (6), and geometric isomers of tambjamine MYP1 ((E/Z)-7) have been developed. The crucial steps involved in these synthetic routes are the construction of methoxy-bipyrrole-carboxaldehydes (MBCs) and a 20-membered macrocyclic core and a regioselective demethylation of MBC analogues. These new synthetic routes enabled us to generate several natural prodiginines 24-27 in larger quantity. All of the synthesized natural products exhibited potent asexual blood-stage antiplasmodial activity at low nanomolar concentrations against a panel of Plasmodium falciparum parasites, with a great therapeutic index. Notably, prodiginines 6 and 24-27 provided curative in vivo efficacy against erythrocytic Plasmodium yoelii at 25 mg/kg × 4 days via oral route in a murine model. No overt clinical toxicity or behavioral change was observed in any mice treated with prodiginines and tambjamines.

Acridones Are Highly Potent Inhibitors of Toxoplasma gondii Tachyzoites.

Acridone derivatives, which have been shown to have in vitro and in vivo activity against Plasmodium spp, inhibit Toxoplasma gondii proliferation at picomolar concentrations. Using enzymatic assays, we show that acridones inhibit both T. gondii cytochrome bc1 and dihydroorotate dehydrogenase and identify acridones that bind preferentially to the Qi site of cytochrome bc1. We identify acridones that have efficacy in a murine model of systemic toxoplasmosis. Acridones have potent activity against T. gondii and represent a promising new class of preclinical compounds.

Lead Optimization of Second-Generation Acridones as Broad-Spectrum Antimalarials.

The global impact of malaria remains staggering despite extensive efforts to eradicate the disease. With increasing drug resistance and the absence of a clinically available vaccine, there is an urgent need for novel, affordable, and safe drugs for prevention and treatment of malaria. Previously, we described a novel antimalarial acridone chemotype that is potent against both blood-stage and liver-stage malaria parasites. Here, we describe an optimization process that has produced a second-generation acridone series with significant improvements in efficacy, metabolic stability, pharmacokinetics, and safety profiles. These findings highlight the therapeutic potential of dual-stage targeting acridones as novel drug candidates for further preclinical development.

Boron Trifluoride Etherate Promoted Microwave-Assisted Synthesis of Antimalarial Acridones.

A microwave-assisted, rapid and efficient method using boron trifluoride etherate (BF3.Et2O) for the synthesis of acridones, via an intramolecular acylation of N-phenylanthranilic acid derivatives, has been developed. The reaction proceeds under solvent-free conditions, tolerates a wide range of functional groups, and provides rapid access to a range of acridones in good to excellent yields. Several of the synthesized acridones exhibited potent antimalarial activities against CQ sensitive and multi-drug resistant (MDR) parasites.

Discovery and Structural Optimization of Acridones as Broad-Spectrum Antimalarials.

Malaria remains one of the deadliest diseases in the world today. Novel chemoprophylactic and chemotherapeutic antimalarials are needed to support the renewed eradication agenda. We have discovered a novel antimalarial acridone chemotype with dual-stage activity against both liver-stage and blood-stage malaria. Several lead compounds generated from structural optimization of a large library of novel acridones exhibit efficacy in the following systems: (1) picomolar inhibition of in vitro Plasmodium falciparum blood-stage growth against multidrug-resistant parasites; (2) curative efficacy after oral administration in an erythrocytic Plasmodium yoelii murine malaria model; (3) prevention of in vitro Plasmodium berghei sporozoite-induced development in human hepatocytes; and (4) protection of in vivo P. berghei sporozoite-induced infection in mice. This study offers the first account of liver-stage antimalarial activity in an acridone chemotype. Details of the design, chemistry, structure-activity relationships, safety, metabolic/pharmacokinetic studies, and mechanistic investigation are presented herein.

MarH, a Bifunctional Enzyme Involved in the Condensation and Hydroxylation Steps of the Marineosin Biosynthetic Pathway.

A novel bifunctional enzyme, MarH, has been identified, and its key functional role in the marineosin biosynthesis successfully probed. MarH catalyzes (1) a condensation step between 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC) and 2-undecylpyrrole (UP) to form undecylprodiginine (UPG) and (2) hydroxylation of the alkyl chain of UPG to form the (S)-23-hydroxyundecylprodiginine (HUPG), which is essential for MarG catalyzed bicyclization toward the formation of an unusual spiro-tetrahydropyran-aminal ring of marineosins. The final enigmatic steps in the marineosin biosynthesis have now been deciphered.

Synthesis and Structure-Activity Relationships of Tambjamines and B-Ring Functionalized Prodiginines as Potent Antimalarials.

Synthesis and antimalarial activity of 94 novel bipyrrole tambjamines (TAs) and a library of B-ring functionalized tripyrrole prodiginines (PGs) against a panel of Plasmodium falciparum strains are described. The activity and structure-activity relationships demonstrate that the ring-C of PGs can be replaced by an alkylamine, providing for TAs with retained/enhanced potency. Furthermore, ring-B of PGs/TAs can be substituted with short alkyl substitutions at either 4-position (replacement of OMe) or 3- and 4-positions without impacting potency. Eight representative TAs and two PGs have been evaluated for antimalarial activity against multidrug-resistant P. yoelii in mice in the dose range of 5-100 mg/kg × 4 days by oral administration. The KAR425 TA offered greater efficacy than previously observed for any PG, providing 100% protection to malaria-infected mice until day 28 at doses of 25 and 50 mg/kg × 4 days, and was also curative in this model in a single oral dose (80 mg/kg). This study presents the first account of antimalarial activity in tambjamines.

Synthesis, structure-activity relationships, and biological studies of chromenochalcones as potential antileishmanial agents.

Antileishmanial activities of a library of synthetic chalcone analogues have been examined. Among them, five compounds (11, 14, 16, 17, 22, and 24) exhibited better activity than the marketed drug miltefosine in in vitro studies against the intracellular amastigotes form of Leishmania donovani. Three promising compounds, 16, 17, and 22, were tested in a L. donovani/hamster model. Oral administration of chalcone 16, at a concentration of 100 mg/kg of body weight per day for 5 consecutive days, resulted in >84% parasite inhibition at day 7 post-treatment and it retained the activity until day 28. The molecular and immunological studies revealed that compound 16 has a dual nature to act as a direct parasite killing agent and as a host immunostimulant. Pharmacokinetics and serum albumin binding studies also suggest that compound 16 has the potential to be a candidate for the treatment of the nonhealing form of leishmaniasis.

Synthesis and insight into the structure-activity relationships of chalcones as antimalarial agents.

Licochalcone A (I), isolated from the roots of Chinese licorice, is the most promising antimalarial compound reported so far. In continuation of our drug discovery program, we isolated two similar chalcones, medicagenin (II) and munchiwarin (III), from Crotalaria medicagenia , which exhibited antimalarial activity against Plasmodium falciparum . A library of 88 chalcones were synthesized and evaluated for their in vitro antimalarial activity. Among these, 67, 68, 74, 77, and 78 exhibited good in vitro antimalarial activity against P. falciparum strains 3D7 and K1 with low cytotoxicity. These chalcones also showed reduction in parasitemia and increased survival time of Swiss mice infected with Plasmodium yoelii (strain N-67). Pharmacokinetic studies indicated that low oral bioavailability due to poor ADME properties. Molecular docking studies revealed the binding orientation of these inhibitors in active sites of falcipain-2 (FP-2) enzyme. Compounds 67, 68, and 78 showed modest inhibitory activity against the major hemoglobin degrading cysteine protease FP-2.